With the lack of new antibiotics in the drug discovery pipeline, coupled with accelerated evolution of antibiotic resistance, a new approach to combating drug resistant Gram-negative pathogens needs to be implemented. Multidrug transporters are a key target in these efforts. In particular, the Resistance-Nodulation-Cell-Division (RND) family of transporters are primarily responsible for the decreased intracellular permeability and retention of antibiotics. We therefore propose to develop new small molecule inhibitors that selectively target RND pumps as adjuvants to antibiotics. Indeed, our long-term goal is to use efflux pump inhibitors (EPIs) in combination with existing antibiotics, to restore antibiotic potency, reverse resistance, and dramatically reduce the rates of resistance development in Gram-negative clinical isolates. Our innovative search for molecules to reverse drug resistance has benefited enormously from understanding the natural history of chemical interactions among organisms, and makes use of our in-house natural product chemical library consisting of over 4,400 crude extracts from marine bacteria, phytoplankton, and fungi. The feasibility of our approach has been established having identified 36 marine isolates able to decrease an antibiotic's MIC by 4-fold or greater from an initial screening of ~1300 crude extracts. Furthermore, we have isolated 3,4-dibromopyrrole-2,5-dione, a new putative EPI from a heterotrophic marine bacteria, which decreased the MICs of seven antibiotics between 2 and 16-fold against strains overexpressing three archetype RND transporters (AcrAB-TolC, MexAB-OprM, and MexXY-OprM), and demonstrated EPI functionality in accumulation/efflux assays. In Aim 1, continued screening efforts against MDR test strains will identify additional marine isolates with antibiotic potentiation capabilities, that will be further tested in dilution series ith and without antibiotics to assess extract synergy. In Aim 2, prioritized leads will be targeted for scaled up production and undergo bio- assay guided fractionation to obtain pure compounds of sufficient quantity for structural elucidation/de- replication and downstream applications. In Aim 3, EPI functionality of lead compounds will be evaluated by accumulation/efflux assays with fluorogenic/radiolabeled compounds. Counterscreens will be performed to rule out protonophores and membrane permeabilizers, and checkerboard assays will be conducted to obtain fraction inhibitory concentrations (FICs) to identify compound-specific synergistic interactions with antibiotics. The R33 phase will be initiated only if our defined milestones are achieved. In Aim 4, compounds meeting selection criteria in the R21 phase will be targeted for micro-scale chemical modification to refine chemical scaffolds based on structure-activity relationship testing, and optimized for minimal toxicity, enhanced potency and solubility. In the final Aim, we will assess in vitro effectiveness of the most promising candidate EPIs in Gram-negative clinical isolates harboring mutations leading to increased synthesis of RND pumps, and establish whether these leads enable killing of MDR pathogens in relevant models of infection.